Ribozymes are biological catalysts consisting of only RNA. They promote a variety of reactions involving RNA and DNA molecules including site-specific cleavage, ligation, polymerization, and phosphoryl exchange (Cech, T. R. [1989] Biochem. Int. 18(1):7-14; Cech, T. R. [1990] Annu. Rev. Biochem. 59:543-569). Ribozymes fall into three broad classes: (1) RNAse P, (2) self-splicing introns, and (3) self-cleaving viral agents. Self-cleaving agents include hepatitis delta virus and components of plant virus satellite RNAs that sever the RNA genome as part of a rolling-circle mode of replication. Because of their small size and great specificity, ribozymes have the greatest potential for biotechnical applications. The ability of ribozymes to cleave other RNA molecules at specific sites in a catalytic manner has brought them into consideration as inhibitors of viral replication or of cell proliferation and gives them potential advantage over antisense RNA. Indeed, ribozymes have already been used to cleave viral targets and oncogene products in living cells (Koizumi, M., H. Kamiya, E. Ohtsuka [1992] Gene 117(2): 179-184; Kashani-Sabet, M., T. Funato, T. Tone et al. [1992] Antisense Res. Dev. 2(1):3-15; Taylor, N. R., J. J. Rossi [1991] Antisense Res. Dev. 1(2):173-186; von-Weizsacker, F., H. E. Blum, J. R. Wands [1992] Biochem. Biophys. Res. Commun. 189(2):743-748; Ojwang, J. O., A. Hampel, D. J. Looney, F. Wong-Stall, J. Rappaport [1992] Proc. Natl. Acad. Sci. USA 89(22):10802-10806; Stephenson, P., I. Gibson [1991] Antisense Res. Dev. 1(3):261-268; Yu, M., J. Ojwang, O. Yamada et al. [1993] Proc. Natl. Acad. Sci. USA 90(13):6340-6344; Xing, Z., J. L. Whitton [1993] J. Virol. 67(4):1840-1847; Yu, M., E. Poeschla, O. YamadaetaL [1995] Virology 206(1):381-386; Little, E., A. S. Lee [1995] J. Biol. Chem. 270(16):9526-9534).
Two kinds of ribozymes have been employed widely, hairpins and hammerheads. Both catalyze sequence-specific cleavage resulting in products with a 5' hydroxyl and a 2',3'-cyclic phosphate. Hammerhead ribozymes have been used more commonly, because they impose few restrictions on the target site. Hairpin ribozymes are more stable and, consequently, function better than hammerheads at physiologic temperature and magnesium concentrations.
A number of patents have issued describing various ribozymes and methods for designing ribozymes. See, for example, U.S. Pat. Nos. 5,646,031; 5,646,020; 5,639,655; 5,093,246; 4,987,071; 5,116,742; and 5,037,746. However, the ability of ribozymes to provide therapeutic benefit in vivo has not yet been demonstrated.
There are more than 200 inherited diseases that lead to retinal degeneration in humans. Considerable progress has been made in identifying genes and mutations causing many forms of inherited retinal degeneration in humans and other animals. Diseases causing inherited retinal degeneration in humans can be classified broadly into those that first affect peripheral vision and the peripheral retina, such as retinitis pigmentosa, and those that primarily affect central vision and the macula, such as macular dystrophy. The macula has the highest concentration of cones and the peripheral retina is dominated by rods.
Retinitis pigmentosa (RP) is a collection of heritable retinal degenerations caused by defects in one of several genes for proteins of photoreceptor(PR) cells. RP is characterized by progressive rod photoreceptor degeneration and eventual blindness. The exact molecular pathogenesis of RP is still unexplained. Ultrastructural observations suggest that the rod PRs are severely affected in the disease. Approximately 50,000 individuals in the United States are estimated to have RP. The clinical symptoms of retinitis pigmentosa include night blindness and loss of peripheral vision. With time visual impairment progresses toward the center of the retina causing "tunnel-vision."
Retinitis pigmentosa can be subdivided into several genetic categories: antosomal dominant (adRP), autosomal recessive (arRP), X-linked (xIRP) or syndromic. There are also a number of clinical classes for retinitis pigmentosa. These classes have been condensed into two broad categories. Type 1 retinitis pigmentosa is characterized by rapid progression and diffuse, severe pigmentation; type 2 retinitis pigmentosa has a slower progression and more regional, less severe pigmentation.
Macular degeneration is a deterioration of the macula (the cone-rich center of vision) leading to gradual loss of central vision. Eventual loss of these cones leads to central vision loss and functional blindness. At least 500,000 individuals are estimated to suffer from macular degeneration currently in the United States. Macular degeneration can have either a genetic basis or it may be an acquired disease. Approximately 10% of Americans over the age of 50 are afflicted with age-related macular degeneration, an acquired form of disease. The inherited forms of macular degeneration are much less common but usually more severe. Inherited macular degeneration is characterized by early development of macular abnormalities such as yellowish deposits and atrophic or pigmented lesions, followed by progressive loss of central vision.
There is currently no effective treatment for most forms of retinitis pigmentosa or macular degeneration. Treatment with a massive supplement (15,000 I.U. per day) of vitamin A often retards the course of retinal degeneration in retinitis pigmentosa. Vitamin therapy does not treat the underlying cause of RP and is not a cure.
There are many other inherited diseases that cause retinal degeneration in humans. Among these are gyrate atrophy, Norrie disease, choroideremia and various cone-rod dystrophies. In addition there are numerous inherited systemic diseases, such as Bardet-Biedl, Charcot-Marie-Tooth,and Refsum disease which include retinal degeneration among a multiplicity of other symptoms.
Another important ocular disease is diabetic retinopathy. Diabetic retinopathy is the leading cause of blindness in adults between the ages of 18-72. Histological studies consistently implicate endothelial cell dysfunction in the pathology.
Hyperglycemia directly contributes to the development of diabetic retinopathy, and early in the development of diabetic retinopathy there exists disruption of the blood-retinal barrier. NOS activity, as determined by conversion of arginine to citrulline, is significantly increased in diabetes Rosen, P., T. M. Danoff, A. DePiero, F. N. Ziyadeh [1995] Biochem. Biophys. Res. Commun. 207(1):80-88). Gade and coworkers demonstrated that endothelial cell dysfunction correlated with elevated glucose in an in vitro wound model and was mediated by increased levels of NO (Gade, P. V., J. A. Andrades, M. E. Nemni et al. [1997] J. Vasc. Surg. 26(2):319-326). In rat cerebral arteries acute glucose exposure dilates arteries via an endothelium mediated mechanism that involves NO (Cipolla, M. J., J. M. Porter, G. Osol [1997] Stroke 28(2):405-411). Cosentino demonstratedthat prolonged exposure to high glucose increases eNOS gene expression, protein synthesis, and NO release Cosentino, F., K. Hishikawa, Z. S. Katusic, T. F. Luscher [1997] Circulation 96(1):25-28).
Nitric oxide (NO) is a pleiotropic molecule with multiple physiological effects: neurotransmitter, component of the immune defense system, regulator of smooth muscle tone and blood pressure, inhibitor of platelet aggregation and a superoxide scavenger. NO is synthesized as a product of the conversion of L-arginine into L-citrulline by the so-called constitutive nitric oxide synthase (NOS), either neuronal (NNOS) or endothelial (eNOS) isoforns. NO regulates specific protein levels. NO increases mRNA levels for VEGF and iNOS.
Although several studies on NO function in the retina have been published, very little information is available pertaining to its role in the diabetic retina (Chakravarthy, U., A. W. Stitt, J. McNally et al. [1995] Curr. Eye Res. 14(4):285-294; Goldstein, I. M., P. Ostwald, S. Roth [1996] Vision Res. 36(18):2979-2974). The iNOS isoform is expressed in the retina, as shown by RT-PCR and immunocytochemistry. It is believed to be involved in the development of diabetic retinopathy and in ischemia-reperfusion injury Hangai, M., N. Yoshimura, K. Hirioi, M. Mandai, Y. Honda [1996] Exp. Eye Res. 63(5):501-509; Ostwald, P., I. M. Goldstein, A. Pachnanda, S. Roth [1995] Invest. Ophthalmol. Vis. Sci. 36(12):2396-2403). Administering NOS inhibitors can ameliorate or prevent ischemia-reperfusioninjury (Lam, T. T., M. O. Tso [1996] Res. Commun. Mol. Pathol. Pharmacol. 92(3):329-340). Diabetic human retinal pigmented epithelial cells have augmented iNOS compared to non-diabetic cells. An increasing body of evidence indicates growth factors including vascular endothelial growth factor (VEGF) and insulin-like growth factor-I (IGF-I) are involved in increased permeability of endothelium that leads to breakdown of the blood-retinal barrier in this microvascular disease. However, the mechanisms for growth factor action in disease progression remain elusive.